Poly(oxy-1,2-ethanediyl), .alpha.-sulfo-.omega.-hydroxy-, C8-10-alkyl ethers, sodium salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Apr - 11 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

adopted in 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certifiying authority: Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Alcohols, C8-10, ethoxylated, sulfates, sodium salts

EC Number:

939-523-2

Cas Number:

1471312-55-6

Molecular formula:

not available

IUPAC Name:

Alcohols, C8-10, ethoxylated, sulfates, sodium salts

Test material form:

semi-solid (amorphous): gel

Remarks:

paste

Method

Target gene:

his-operon

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

other: All strains are deep rough and have a reduced capability to repair DNA-damage (except for TA 102). Strains TA 98, TA 100 and TA 102 contain the R-factor plasmid pkM101 enhancing an error prone DNA repair system.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix prepared from the livers of rats treated with phenobarbital/beta-naphthoflavone was achieved from Trinova Biochem GmbH and was cofactor supplemented.

Test concentrations with justification for top dose:

plate incorporation and preincubation test: 3.16, 10.0, 31.6, 100, 316, 1000, 2500, 5000 µg/plate

Vehicle / solvent:

- Vehicle used: distilled water

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

A. dest.

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD) and 2-Aminoanthracene (2-AA)

Details on test system and experimental conditions:

A preliminary plate incorporation experiment with the tester strains TA 98 and TA 100 was performed. The results were were in accordance with the acceptance criteria and are therfore reported as part of Experiment I.

METHOD OF APPLICATION:
Experiment I: in agar (plate incorporation);
Experiment II: preincubation

The initial plate incorporation test was conducted as follows:
Briefly, 0.1 mL test substance, 0.1 mL bacteria culture, 0.5 mL S9 mix (or buffer) and 2.0 mL soft agar were mixed and plated onto petri dishes with solid agar. After incubation at 37 °C for 48 h colonies were counted using an automatic counter. Each experiment was performed in triplicate.
The independent repeat was performed as preincubation test. Briefly, 0.1 mL test substance, 0.1 mL bacteria culture and 0.5 mL S9 mix (or buffer) were preincubated at 37 °C for 60 min. At the end of the preincubation period 2 mL of molten soft agar was added. After mixing, the suspension was plated, incubated for 48 h at 37 °C and colonies were counted using an automatic counter. Each experiment was performed in triplicate.

DETERMINATION OF CYTOTOXICITY
The reduction of background growth of bacteria on the plates as well as a reduction in the mutant count per plate to approximately 0.5 in comparison to control were used as marker for cytotoxicity.

Evaluation criteria:

A clear and dose-related and/or biologically relevant increase in mutant counts for at least one strain is considered to be a positive result. For TA 98, TA 100, and TA 102 this increase should be about twice that of negative controls. For TA 1535 and TA 1537 the increase should be about three times than that of solvent controls. Otherwise, the result is evaluated as negative. However, these criteria may be overruled by good scientific judgment.

Statistics:

Mean values and standard deviation were calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. I: at ≥ 2500 µg/plate; -S9 Exp. I: at 5000 µg/plate; + S9 Exp. II: at ≥ 1000 µg/plate; -S9 Exp. II: at ≥ 2500 µg/plate; +S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. I: at ≥ 2500 µg/plate; -S9 Exp. II: at 316 µg/plate; -S9 Exp. II: at 2500 µg/plate; +S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. I: at ≥ 2500 µg/plate; -S9 Exp. II: at ≥ 1000 µg/plate; -S9 Exp. II: at ≥ 2500 µg/plate; +S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. I: at ≥ 1000 µg/plate; -S9 Exp. I: at 5000 µg/plate; +S9 Exp. II: at ≥ 1000 µg/plate; -S9 Exp. II: at ≥ 2500 µg/plate; +S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. II: at 5000 µg/plate; -S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No precipitation was observed in any tester strain at any concentration.

Results of the preliminary experiment are found under Table 1 (Any other information on results incl. tables).

Any other information on results incl. tables

Table 1: Test results of the dose-setting study

Substance	Dose (µg/plate)	TA 98 Mutation Factor [toxicity]*   Without S9	TA 98 Mutation Factor [toxicity]*     With S9	TA 100 Mutation Factor [toxicity]*     Without S9	TA 100 Mutation Factor [toxicity]*   With S9
Solvent Control (A. dest)		1.0	1.0	1.0	1.0
4-NOPD	10.0	18.5	-	-	-
NaN3	10.0	-	-	8.7	-
2-AA	2.50	-	75.4	-	12.7
Test item	3.16	1.1	1.3	1.2	1.0
	10.0	0.8	1.3	1.3	1.0
	31.6	1.0	1.5	1.3	0.9
	100	1.1	1.4	1.1	1.1
	316	1.1	1.2	0.9	1.0
	1000	0.8	1.4	0.7B	0.9
	2500	0.7B	1.2	0.4B	0.8
	5000	0.6B	0.8	0.0B	0.5B

* [toxicity paramter]: B=Background lawn reduced

 

Table 2: Test results of experiment 1 (plate incorporation).

Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
	Base-pair substitution type			Frameshift type
	TA 100	TA1535	TA102	TA98	TA1537
without S9-Mix
0	107 ± 7.5	6 ± 3.1	175 ± 10.6	25 ± 10.4	9 ± 2.5
3.16	128 ± 14.6	10 ± 6.7	196 ± 13.1	27 ± 6.4	14 ± 4.5
10.0	134 ± 4.2	7 ± 0.6	174 ± 13.4	21 ± 3.1	12 ± 3.1
31.6	134 ± 17.3	8 ± 1.7	185 ± 25.1	25 ± 2.5	9 ± 0.6
100	120 ± 15.0	8 ± 0.0	164 ± 15.1	28 ± 7.5	12 ± 2.5
316	95 ± 8.5	10 ± 3.2	182 ± 15.1	27 ± 9.7	8 ± 2.5
1000	74 ± 13.2 B	10 ± 4.2	215 ± 10.8	21± 3.1	9± 4.2
2500	38 ± 3.0 B	0± 0.0 B	179± 10.8	19± 6.5 B	6± 4.6 B
5000	0± 0.6 B	0± 0.0 B	148± 11.0	14± 2.5 B	0± 0.0 B
Positive controls:	Na-azide		MMS	4-NOPD
Concentration (μg/plate)	10		1 µL/plate	10	40
Mean No. of colonies/plate (average of 3 ± SD)	934 ± 22.0	758 ± 220.3	1782 ± 345.7	461 ± 23.8	119 ± 7.8
With S9-Mix
0	124 ± 13.8	7 ± 4.0	253 ± 5.3	26 ± 1.0	7 ± 3.5
3.16	120 ± 20.6	7 ± 7.1	272 ± 21.0	35 ± 5.2	11 ± 2.5
10.0	120 ± 8.1	10 ± 3.6	262 ± 2.5	35 ± 2.9	13 ± 4.5
31.6	113 ± 12.5	10 ± 3.2	262 ± 7.2	40 ± 7.2	10 ± 3.5
100	133 ± 16.7	7 ± 1.7	248 ± 19.7	35 ± 7.6	9 ± 1.0
316	121 ± 4.6	9 ± 1.0	277 ± 7.8	32 ± 3.5	12 ± 1.2
1000	117 ± 29.5	11 ± 1.7	274 ± 7.0	36 ± 5.3	13 ± 3.8
2500	100 ± 9.9	7 ± 1.5	233 ± 8.4	31 ± 3.1	6 ± 2.5
5000	61 ± 4.4 B	4 ± 1.0 B	238 ± 28.6	20 ± 5.5	9 ± 1.5
Positive controls:	2-AA
Concentrations (μg/plate)	2.5		10	2.5
Mean No. of colonies/plate (average of 3 ± SD)	1581 ± 229.7	102 ± 9.0	516 ± 44.6	1959 ± 504.5	261 ± 14.9

MMS = Methylmethanesulfonate

4-NOPD = 4-Nitro-o-phenylene diamine

2-AA = 2-Aminoanthracene

B = Reduction of background lawn

 

 

Table 2: Test results of experiment 2 (pre-incubation).

Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
	Base-pair substitution type			Frameshift type
	TA 100	TA1535	TA102	TA98	TA1537
without S9-Mix
0	115± 23.1	10± 4.0	270± 12.2	22± 3.2	7± 1.5
3.16	129± 14.4	4± 2.1	241± 29.2	24± 1.2	10± 2.0
10.0	133± 9.0	9± 2.3	264± 29.4	31± 2.5	10± 6.0
31.6	119± 4.4	5± 1.0	278± 29.7	23± 2.6	12± 6.4
100	114± 15.9	8± 2.0	283± 23.1	26± 1.0	9± 3.2
316	100± 6.1	7± 1.2	262± 9.8	25± 2.1	8± 1.2 B
1000	58± 9.1 B	0± 0.0 B	280± 22.8	25 ± 0.6 B	0 ± 0.0 B
2500	0 ± 0.0 B	0 ± 0.0 B/N	155 ± 37.0	22 ± 6.1 B	0 ± 0.0 N
5000	0 ± 0.0 N	0 ± 0.0 N	125 ± 45.0 B	0 ± 0.0 B	0 ± 0.0 N
Positive controls:	Na-azide		MMS	4-NOPD
Concentration (μg/plate)	10		1 µL/plate	10	40
Mean No. of colonies/plate (average of 3 ± SD)	965± 120.2	1038± 227.1	2158± 67.5	389± 30.1	107± 7.0
With S9-Mix
0	108± 12.7	10± 2.5	292± 7.8	38± 4.5	9± 3.2
3.16	137± 30.7	5± 2.1	289± 15.9	39± 5.6	7± 2.1
10.0	139± 4.2	5± 2.5	323± 36.5	37± 2.0	4± 2.9
31.6	121± 14.0	7± 3.1	310± 21.2	32± 4.5	5± 4.9
100	134± 14.7	7± 1.5	297± 6.1	31± 6.0	7± 2.6
316	116± 7.6	8± 1.5	286± 24.0	31± 6.7	4 ± 1.2
1000	101± 15.5	7± 0.6	310± 7.4	30± 12.7	12± 3.5
2500	85± 11.8 B	4± 2.5 B	284± 15.0	22± 4.2 B	7± 3.5 B
5000	70± 4.6 B	0± 0.0 B	273± 10.7	21± 10.4 B	6± 3.6 B
Positive controls:	2-AA
Concentrations (μg/plate)	2.5		10	2.5
Mean No. of colonies/plate (average of 3 ± SD)	789± 158.7	60 ± 15.0	628 ± 14.3	2943 ± 399.9	128 ± 29.8

MMS = Methylmethanesulfonate

4-NOPD = 4-Nitro-o-phenylene diamine

2-AA = 2-Aminoanthracene

B = Reduction of background lawn

N = No background lawn

Applicant's summary and conclusion

Conclusions:

From the results in this study and under these test conditions, the test substance was assessed not to be mutagenic.